Intracellular Accumulation of Lipids via Scavenger Receptors
Qi Chen
Nanjing Medical University
2005.8
Formation of foam cell
Athersclerosis is a chronic inflammatory reaction caused by macrophages and lymphocytes that uptake the pathogenicity lipids invading the vessel.
SR-A and CD36 are the principal receptors responsible for foam-cell formation
Macrophage cell takes up lipid by SR-A
Structure of SR-A
Six domains
Three types
Trimeric molecule
Foam cell formation
Mφ adhesion
Host defense and innate
immunity
Adaptive responses
Disorders of the nervous
system
Phagocytosis of apoptotic
cells
Functions of SR-A
The cytoplasmic domain is the key part of the receptor???
mediate the internalization of ligands such as ox-LDL
participate the regulation of cell adhesion and migration
accommodate the expression and transport of receptor
link-coupled with downstream of signaling pathway
Functions of SR-A cytoplasmic domain:
Cytoplasmic domain：
Murine: MTKEMTENQRLCPHEREDADCSSESVKFDARSMTASLP
HSTKNGPSVQEKLKSFK (55)
Rabbit: MAQWDSFTDQQEDTDSCSESVKFDARSNTALLPPNPK
GPPLQEKLKSFK (50)
Bovine: MAQWDDFPDQQEDTDSCTESVKFDARSVTALLPPHPKN
GPTLQERMKSYK (50)
Human: MEQWDHFHNQQEDTDSCSESVKFDARSMTALLPPNPK
NSPSLQEKLKSFK (50)
phosphorylation of cytoplasmic domain
signal sequence in cytoplasmic domain
interactors with cytoplasmic domain
Focus on cytoplasmic domain：
Bovine Model
Human Model
Murine Model
Rabbit Model
Ser21（rat）
increases expression of receptor
promotes metabolism of lipid
Fong LG et al. J Biol Chem, 274, 36088-36816 (1999)
phosphorylation of cytoplasmic domain
phosphorylation of cytoplasmic domain
Ser49（rat）
reduces uptake of lipid
Fong LG et al. J Biol Chem, 274, 36088-36816 (1999)
FDARS
internalization of receptor
Fong LG et al. J Biol Chem, 274, 36088-36816 (1999)
signal sequence of cytoplasmic domain
KLKSFK
cell adhesion
Kosswig N et al. J Biol Chem, 278, 34219-34225 (2003)
signal sequence of cytoplasmic domain
VXFD (rabbit)
internalization of receptor, transference to cell surface
Morimoto K et al.Biol Pharm Bull, 22, 1022-1026 (1999)
signal sequence of cytoplasmic domain
Ser48 (human）
expression and internalization of receptor, cell migration
Heider H et al. FEBS Letters, 505, 185-190 (2001)
phosphorylation of cytoplasmic domain
Di-leucine motif ?
signal sequence of cytoplasmic domain
1. The role of Di-leucine motif in the cytoplasmic domain of SRA
(1) SR-A mutants/EGFP constructs:
Wild type
N3132LM
EGFP
SRA
SRA
(2) Expression patterns of SRA mutant:
*P<0.05,n=3
EGFP
SR-A N3132LM
EGFP
SR-A
N3132LM
*
0
200
400
600
800
1000
control
EGFP
SR-A
N3132LM
(3) The internalization of DiI-AcLDL via SRA mutant:
DiI-acLDL
SR-A expression
*
*
DiI-acLDL
*P<0.05,n=3
* *P<0.05,n=3
0
10
20
30
40
EGFP
Wild type
N3132LM
*
EGFP
Wild type
N3132LM
0.02
0.04
0.06
0.08
0.10
0.12
(4) Cell adhesion via SRA mutant:
*
*P<0.05, n=3
*
EGFP
SR-A
N3132LM
Immunofluorescence: The CHO cells were transfected with SRA/EGFP or SRA Mutant/EGFP,
(5) Di-leucine motif and Clathrin-coated pit:
GFP
Anti-clathrin
overlay
180KD
97KD
a b c
a: EGFP-transfected cell
b: SR-A-transfected cell
c: N3132LM mutant-transfected cell
Clathrin IP:
NΔ1-27
NΔ1-27LM
*
*
*P<0.05,n=3
SR-A NΔ1-27 NΔ1-27LM
97KD
A
B
C
(6) Expressing of the SRA mutants in CHO cell
EGFP
SR-A
400
600
800
1000
NΔ1-27
NΔ1-27LM
200
(7) The internalization of DiI-AcLDL via SRA mutant:
*P<0.05,n=3
*P<0.05,n=3
*
*
*
*
DiI-acLDL
DiI-acLDL
SR-A expression
10
20
30
40
EGFP
SR-A
NΔ1-27
NΔ1-27LM
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0
EGFP
SR-A
NΔ1-27
NΔ1-27LM
* Di-leucine signal motif in the cytoplasmic domain is required for MSR-A-mediated internalization.
* Di-leucine signal motif-related endocytosis of Ac-LDL may depend on the clathrin-coat pit.
* There might be multiple signal motives in cytoplasmic domain of MSR, which adapts the multiple functions of MSR. Di-Leucine motif may be an distinct motif from the VXFD in SR-A mediated internalization and adhesion.
2. Identification of peptide ligands for the cytoplasmic domain of SRA
(1) GST -cSRA fusion protein expression and purification:
GST fusion protein were separated on a 12% SDS-PAGE. 1, purified GST fusion protein; 2, E. coli supernatant containing GST fusion protein; 3 and 6, E. coli supernatant; 4, molecular weight standards; 5, E. coli supernatant containing GST.
1 2 3 4 5 6
Western-blot of the fusion protein with
anti-GST antibody. 1, GST-cytoplasmic domain
of SRA fusion protein;2, GST; 3, E. coli supernatant;
4, molecular weight standards.
1 2 3 4
(2) Screening the phage peptide library with the fusion protein :
The pool of phage becomes enriched in favour of sequences that bind to the target after four
rounds of bio-panning
* phage form unit
** output phages/input phages
Binding specificity of the positive phage clones analysed by ELISA using anti-M13 antibody. The black, binding with the fusion protein (P); The white, binding with GST (N). The positive clones : P/N>3.
(3) Isolation of binding phage clones by Phage ELISA:
II4 II5 II6 II7 II9 II10 II11 II12 M13
(4) Sequencing the selected positive clones:
* H11: TDRLFMNSIWPG (4, 6, 7, 10, 11, 12)
* H5: TFWDDGWLLLPR (5)
* H9: YGDRWFMPVMRS (9)
(5) Searching the PIR-PSD database :
H5: Casein kinase II phosphorylation site (TFWD)
H11:Protein kinase C phosphorylation site (TDR)
(6) Construction of expression plasmids
PEGFP-N1-H5
PEGFP-N1-H9
PEGFP-N1-H11
EGFP
P